Evaluation of candidate reference genes for gene expression analysis in the brassica leaf beetle, Phaedon brassicae (Coleoptera: Chrysomelidae)

The brassica leaf beetle Phaedon brassicae is a notorious defoliator of cruciferous vegetables. However, few molecular studies of this pest have been conducted due to limited sequence data. Recently, RNA sequencing has offered a powerful platform to generate numerous transcriptomic data, which require RT-qPCR to validate target gene expression. The selection of reliable reference genes to normalize RT-qPCR data is a prerequisite for gene expression analysis. In the present study, the expression stabilities of eight candidate reference genes under biotic conditions (development stages and various tissues) and abiotic perturbations (thermal stress and pesticide exposure) were evaluated using four different statistical algorithms. The optimal suites of reference genes were recommended for the respective experimental conditions. For tissue expression analysis, RPL32 and EF-1α were recommended as the suitable reference genes. RPL19 and TBP were the optimal reference genes across different developmental stages. RPL32 and TBP were identified as the most suitable references for thermal stress. Furthermore, RPL32 and RPL19 were ranked as the best references for insecticide exposure. This work provides a systematic exploration of the optimal reference genes for the respective experimental conditions, and our findings would facilitate molecular studies of P. brassicae.

of validating reference genes in different conditions, in order to obtain a set of standard genes that work best for appropriate conditions. The ms is well written, and analyses seem appropriate. line 116: ..70% ± 10,… line 126-129: how were the insects exposed, by feeding, filter paper petri dish, injection? Please clarify.
Respond: Thanks for your suggestion. In this work, the insecticide exposure experiment was performed following a leaf-dipping method (Cui et al., 2018;Lutz et al., 2018). In the present version, we make a detailed description as follows, 'the leaf discs of radish (2 cm diameter) were dipped in the solutions with insecticide for 10 s, then left to air-dry at room temperature. Each piece of the dipped leaf discs was placed in a Petri dish, into which four larvae were transferred'.   Respond: Thanks for your suggestion. In the section of Results, the best reference genes were recommended for each experimental conditions as follows, 'Integrating the evaluation of all four programs, the comprehensive ranking of candidate reference genes was determined by RefFinder (Table   2), wherein RPL19 and TBP were considered as the best reference genes across different developmental stages, RPL32 and Ef-1α were recommended for tissue expression analysis, RPL32 and TBP were identified as the optimal candidates for the temperature experiment, and RPL32 and RPL19 were selected for the insecticide treatment'.

Reviewer #2: Authors Ma et al. performed a study to evaluate candidate reference genes for molecular studies in Brassica Leaf Beetle, a serious pest of brassicaceous vegetables in East and South
Asia. The research is important as identifying the most stable reference genes for a species is required for many molecular research based on the quantitative real-time PCR technique. In general, the experimental design and data analysis of this study are scientifically sound. However, the clarification of data presentation, language quality, discussion and citation need to be enhanced significantly.
Following are some of my specific suggestions.
1. The English of this manuscript should be polished considerably. There are many errors (spelling, grammar, etc.) and clarification problems. Authors should rewrite some sentences to make it clear.
Honestly speaking, asking for editorial help from a native speaker will be optimal.
Respond: Thanks for your constructive suggestion, and we check our writing thoroughly to prevent the misleading expression.

Title: Evaluation, not Evalution
Respond: Thanks for your correction.
1.2. Title: change "in a Brassica Leaf Beetle" to "in the Brassica Leaf Beetle" Respond: Thanks for your correction.
1.4. Rewrite the sentence in Lines 54-57. Two sentences instead of one will be ideal. Using However instead of but Respond: Thanks for your suggestion. In the current version, we cited more references to describe the field management against P. brassicae as follows, 'however, the application of insecticides was not always effective against P. brassicae due to its complex lift history and high fecundity (Wang et al., 1.11. Line 214: change "Wih" to "With" Respond: Thanks for your correction. In the present version, this was corrected to 'in regards to'.
1.13. Line 230: add "ones" after "RPL19 as the most stable" Respond: Thanks for your suggestion.
1.14. Lines 225-226: change this sentence to "ranked RPL32, TBP and α-TUB as the top three reference genes" Respond: Thanks for your correction. In the present version, this sentence was corrected to 'NormFinder and geNorm ranked RPL32, TBP and α-TUB as the top three reference genes'.
1.15. Line 227: add "the" -in "the" pesticide exposure experiment Respond: Thanks for your correction. Respond: Thanks for your suggestion. In the section of 'Insect Rearing', we made a description as follows, 'This strain has been reared for six generations without exposure to chemical insecticides'.

Line 143-please provide the accession numbers of these candidate reference genes in NCBI database
Respond: Thanks for your suggestion. In the current version, the sequences and accession numbers of the candidate reference genes have been supplemented in Table 1 and Table S1. Respond: Thanks for your suggestion. In the end of this paragraph, we described how the comprehensive ranking of candidate reference genes was determined by RefFinder as follows, "Finally, a comprehensive analysis tool RefFinder (https://www.heartcure.com.au/reffinder/#) was used to integrate results of the four different analytical methods and assess the rank of reference genes based on their geometric mean". Furthermore, these references were cited as suggested.  Table 1. Please provide the accession numbers for these candidate genes Respond: Thanks for your suggestion. In the current version, the accession numbers were supplemented in Table 1, and meanwhile the sequences of the eight candidate reference genes were added in supplementary data (Table S1) Respond: Thanks for your suggestion. In the section of Materials and methods, we described how the comprehensive ranking of candidate reference genes was determined by RefFinder as follows, "Finally, a comprehensive analysis tool RefFinder (https://www.heartcure.com.au/reffinder/#) was used to integrate results of the four different analytical methods and assess the rank of reference genes based on their geometric mean". Similarly, in the Discussion section, please cite more papers, especially some review or representative ones (e.g. insect pests, beneficial insects, mites, other arthropods or animals, plants, etc.) that have summarized the key questions in this research topic. Otherwise, this research is just simply mimicking previous publications in a different species! Respond: Thanks for your constructive suggestion. In the current version, more references were employed to consolidate our arguments, and we rewrote the last paragraph to deepen our discussion, according to your comments.

Reviewer #3:
The authors evaluated the potential of 8 genes as the reference gene that could be used in RT-qPCR assay. Four algorithms were applied to score each candidate gene across four types of samples and yielded 4 suites of optimal reference genes combination for each of the detected 4 given type of samples. The experiments were carefully designed and the results are reliable. This work is important to support the future gene functional study of Phaedon brassicae. The ms was carefully written with a clear logic flow. However, I would advise authors to get this manuscript checked by native English speaker. I believe this manuscript is a good piece of work but also has lot of scope for English language corrections. No major problem could be found. But some minor revisions need to be made before publication.